A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis

Smith‐Lemli‐Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus. The role of cholesterol in Hh protein biogenesis suggested that impaired Hh autoprocessing might underlie some of the developmental abnormalities in SLOS (Fig. 1 and Table 1; ref. 1). An additional role for cholesterol in Hh signal response was suggested by the observation that cyclopamine and jervine, teratogenic plant alkaloids that block Hh signaling, also inhibit cholesterol transport and synthesis 2,3 . But cyclopamine has since been shown to specifically inhibit Hh signaling by binding to a pathway component 4 , and the doses of these alkaloids required to inhibit Hh signaling are lower than those required to block cholesterol transport (ref. 5 and M.K.C., unpublished data). To determine how cholesterol may affect Hh signaling in embryonic development, we exposed chick embryos to cyclodextrin, a cyclic oligosaccharide that forms non-covalent complexes with sterols 6 and can be used to extract and deplete cholesterol from living cells 7 . Cyclodextrin treatment caused variable loss of the frontonasal process and other midline structures (Fig. 2a), and the spectrum of facial defects was similar to that resulting from exposure to the Hh-pathway antagonist jervine 3 . The most severely affected embryos developed a proboscis-like structure that phenocopies the nasal rudiments of mouse embryos that are homozygous with respect to mutations in the gene Sonic hedgehog (Shh; ref. 8).